Gonadectomy and reduced physical activity: Effects on skeletal muscle

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Gonadectomy and Reduced Physical Activity: Effects on Skeletal Muscle Marybeth Brown, PhD, PT, Jonathan S. Fisher, PhD, Eileen M. Hasser, PhD ABSTRACT. Brown M, Fisher JS, Hasser EM. Gonadectomy and reduced physical activity: effects on skeletal muscle. Arch Phys Med Rehabil 2001;82:93-7.

cine and the American Academy of Physical Medicine and Rehabilitation

Objectives: To examine the effects of testosterone (TST) loss on skeletal muscle contractile function and the potential interactive effects of TST loss and physical inactivity. Design: Randomized control trial. Animals: Forty-eight male rats (age, 6mo) were placed into control (Con) or gonadectomized (Orx) groups. Intervention: Two weeks after Orx or sham surgery, half the Con and Orx rats were hind-limb unloaded (HLU) to reduce muscle activity for 2 weeks. Subsequently, in situ contractile function tests were performed on the soleus (SOL), plantaris (PLAN), peroneus longus (PER), and extensor digitorum longus (EDL). These 4 muscles and gastrocnemius (GAST) then were removed, weighed, sectioned, and stained with adenosine triphosphatase for fiber typing and fiber area measures. Main Outcome Measures: Peak tetanic tension (P0), time to peak twitch contraction (TPT), half relaxation time (RT1/2), muscle mass, fiber area, and specific tension (ratio of P0/muscle mass). Results: Body weight and muscle mass were similar in the Con and Orx groups. The ratio of P0 to muscle mass was significantly ( p ⬍ .05) reduced with Orx in SOL (20%), PLAN (18%), PER (28%), and EDL (20%). TPT and RT1/2 were significantly faster after Orx in PLAN, PER, and EDL. HLU significantly reduced muscle mass in SOL, PLAN, and GAST in Orx and intact groups. HLU also caused a significant decline in SOL and PLAN P0. The loss in P0 in the Orx-HLU rats was no greater than the decline in P0 with HLU alone. Conclusions: Gonadectomy results in a loss of P0 regardless of muscle fiber type or function, it is likely to speed up TPT and RT1/2, and it does not exacerbate HLU-related atrophy and P0 loss. Findings may have implications for men with reduced TST levels, as in aging, for instance. Key Words: Muscle contraction; Muscle fibers, fast-twitch; Muscle fibers, slow-twitch; Rats, Sprague-Dawley; Rehabilitation; Testosterone. © 2001 by the American Congress of Rehabilitation Medi-

RELATIONSHIP BETWEEN testosterone (TST) and A muscle mass and strength has been apparent for many years. For example, in maturing animals, an increase in cir-

From the Program in Physical Therapy, Washington University School of Medicine, St Louis, MO (Brown, Fisher); and the Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, MO (Hasser). Accepted in revised form April 21, 2000. Supported by the National Institute on Aging (National Institute of Health [NIH]; grant nos. AG 15796, AG 00585); the National Center for Medical and Rehabilitation Research (NIH; grant no. HD 07434); and the Heart and Lung Institute (NIH; grant no. HL 55306). No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the author(s) or upon any organization with which the author(s) is/are associated. Reprint requests to Marybeth Brown, PhD, PT, Washington University Schl of Medicine, Program in Physical Therapy, Box 8502, 4444 Forest Park Blvd, St Louis, MO, 63108, e-mail: [email protected]. 0003-9993/01/8201-5808$35.00/0 doi:10.1053/apmr.2001.18697

1

culating levels of TST is associated with a gain in muscle size and strength.2 The converse, a loss in strength and muscle mass, occurs when physiologic serum levels of TST are reduced,3,4 as happens, for instance, after gonadectomy. Recent data suggest a differential effect of TST on muscles with varying fiber types and function. Sidor and Blackburn5 showed that leopard frogs’ sexual dimorphism is correlated with TST sensitivity. The frogs’ large, well-developed elbow flexors were highly responsive to TST, but their elbow extensors, which show little dimorphism, were not. Bricout et al6 found that steroid receptors in fast-twitch muscles had a higher TST affinity than those in slow-twitch muscles. These findings suggest that reduced physiologic levels of TST may affect muscle mass and strength differentially, depending on fiber type and perhaps muscle function. Hind-limb unweighting (HLU) is an animal model of reduced physical activity.7 Like other forms of reduced activity (eg, bed rest, space flight), unweighting produces a rapid decline in muscle mass and strength, especially in the weightbearing and locomotor musculature.8,9 Previous work10 from our laboratory showed significant (16%–26%) muscle weight loss in the weight-bearing or locomotor soleus (SOL), plantaris (PLAN), and gastrocnemius (GAST) muscles with a 2-week period of HLU. No change in mass was seen in the non– weight-bearing extensor digitorum longeus (EDL) and a nonsignificant (8%) change was seen in peroneus longus (PER).10 Occurring concomitantly during HLU in male rats is a significant drop in circulating TST levels, leading to the speculation that TST supplementation would prevent the HLU-associated loss in muscle mass.11 Wimalawansa et al12 reversed the HLUinduced loss in muscle volume in TST-treated rats. Twelve days of unweighting without TST supplementation resulted in a 28.5% loss in total body muscle volume as assessed by magnetic resonance imaging, whereas rats treated with TST during the unweighting period had no decline in muscle volume.12 However, contrary to this finding, Bricout6 did not find TST to prevent HLU-induced atrophy in the SOL muscle. Male rats were given either TST or placebo during a 3-week period of HLU, and the amount of SOL atrophy after unweighting was similar in the placebo and TST-supplemented groups. The different outcomes for these 2 studies support the possibility of a differential response of muscles to TST. Possibly, muscles with a high proportion of fast-twitch fibers responded to TST and such muscles as the SOL, with a high percentage of slow-twitch fibers, did not respond to TST. In the aforementioned studies,11,12 contractile muscle function was not examined. Both HLU and gonadectomy (Orx) result in a loss of muscle mass and force, with some evidence indicating that the loss in muscle force is greater than the muscle mass loss.13,14 The combined effects of unweighting and Orx, particularly on muscle contractile function, are not Arch Phys Med Rehabil Vol 82, January 2001

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MUSCLE RESPONSE TO GONADECTOMY AND REDUCED USE, Brown

known. The primary purpose of the present study was to examine further the effects of Orx on extremity skeletal muscle function, specifically contractile force. A second objective was to examine the potential interaction of TST loss and inactivity. Given the decline in TST with aging and the tendency for older men to be very inactive, this study was intended as a first step in developing a greater understanding of age- and inactivityrelated changes in muscle strength. Strength losses, particularly in the older adult, are associated with functional decline, such as rising from a chair, stair climbing, and walking.15 Thus, strategies such as TST hormone replacement to optimize strength may be needed to maintain physical independence in old age. METHODS Animals Virus-free Sprague-Dawley rats 6 to 7 months of age were obtained from Charles River Laboratories.a We chose mature rats to remove rapid growth as a possible confounding factor in the experimental design. On receipt, rats were placed in a virus-free facility, 2 per cage, and maintained in a barrier room where temperature (from 22°–23°C) and hours of light and dark were controlled (12hr/12hr light/dark). Food and water were available ad libitum. After 7 to 10 days of acclimatization to the animal facility, rats were placed randomly into 1 of 4 groups: control (Con, n ⫽ 14), gonadectomized control (Orx, n ⫽ 15), HLU (n ⫽ 9), and gonadectomized-HLU (OrxHLU, n ⫽ 10). Only 10 animals were unweighted, because pilot data using muscle force and contraction time as variables indicated that a sample size of 8 rats would provide adequate power to address the questions of interest. Gonadectomy After administering an intraperitoneal injection (80␮L/100g body weight) of ketamine hydrochloride (75mg/mL) and promazine hydrochloride (12.5mg/mL), we shaved the surgical site and cleansed it thoroughly with povidone-iodine (Betadine威). A midline scrotal incision was made, and the junction between each testicle and spermatic duct was located and tied with 2.0 silk. Testes were removed bilaterally. In the case of sham surgery, a midline incision was made and subsequently sutured. Rats were allowed 2 weeks to recover from the surgery; during that time, they were monitored to ensure that they were eating properly, gaining weight, and moving about normally. Hindlimb unloading Two small casts were applied to HLU animals, 1 around the tail and the other around the upper thorax, to prevent animals from chewing the tail cast. Wires from the tail and thorax were fed through a leader that was hooked to a wire at the top of the cage and spanned the length of the 45 ⫻ 45cm Plexiglass cage. Animals could gain access to the entire enclosure by using their forelimbs. To acclimatize them to the unloading apparatus, we placed rats in the HLU and Orx-HLU groups in the apparatus for 1 hour daily for 5 days before the 2 weeks of unloading. Concurrently, Con and Orx animals were placed in single cages for 1 hour daily for 5 days and then housed individually for 2 weeks while the other animals underwent hindlimb unloading. By the time animals acclimatized to the new animal facility, recovered from surgery, accommodated to the single cages, and underwent 2 weeks of unloading or normal cage activity, they were between 7 and 8 months of age. All protocols were approved by the animal studies committees of both Washington University School of Medicine and the University of MissouriArch Phys Med Rehabil Vol 82, January 2001

Columbia. All procedures met recommendations for animal care published by the American Physiological Society. Contractile Properties Rats were anesthetized with sodium pentobarbital (50mg/kg body weight), and anesthesia was maintained with an additional 2.5–5-mg/kg dose given every 45 to 60 minutes. Body temperature was maintained by keeping the rats on a 39°C water-jacketed heating pad. The SOL, PLAN, EDL, and PER were surgically exposed at their insertion points. The distal end of each tendon was then attached with 2.0 silk to a Grass force transducer,c and the exposed portions of the muscles were bathed with 37°C rat Ringer solution. SOL (postural antigravity, slow twitch), PLAN (locomotor, fast-twitch type IIb ⬎ IIa), PER (locomotor, fast-twitch type IIa ⬎ IIb), and EDL (nonpostural, fast twitch) were selected to permit study of muscles with different proportions of fast and slow fiber types and different anatomic functions.10,16 The tibial and peroneal nerves were isolated, placed on a bipolar stimulating electrode, and covered with 37°C mineral oil. The left hindlimb was rigidly immobilized. Before contractile properties were obtained, animals were allowed to thermoequilibrate for about 30 minutes. We studied the muscles in the following order: PLAN, SOL, PER, and EDL. A length-tension curve for each muscle was obtained, and muscles were adjusted to optimal length. We obtained peak isometric twitch tension with supramaximal 0.5-ms square-wave pulses,b and we determined peak isometric twitch tension, time to peak twitch tension (TPT), and one half relaxation time (RT1/2). Peak tetanic tension (P0) was elicited by 0.5-ms supramaximal pulses delivered at 100Hz (duration, 400ms) for SOL and at 150Hz for EDL (duration, 250ms), PLAN (duration, 300ms), and PER muscles (duration, 350ms). For each muscle, the adjustment to optimal length and the subsequent measurement of P0 were performed only once. Several minutes passed between tests of muscles innervated by the same nerves (PLAN/SOL, PER/EDL), so fatigue probably was not a factor in measurement of P0. (P0 has been found in our laboratory to be highly reproducible.10,20) Histology After contractile properties were obtained, the SOL, EDL, PER, PLAN, and GAST muscles were removed and weighed. GAST was included because of its locomotor importance and size (largest calf muscle), and because it is highly affected by unloading.16,17 Muscles were pinned at their in situ length, embedded in ornithine carbamoyl transferase tissue-freezing medium, frozen in liquid nitrogen, and placed in a freezer at ⫺80°C until analysis. Muscles were sectioned at 8␮m2, preincubated at 4.3 and 4.55pH, and incubated for adenosinetriphosphatase (ATPase) activity for determination of major fiber types I, IIa, and IIb.18 Fiber typing was done to determine if changes in contractile function (eg, TPT) were related to changes in fiber type. ATPase-stained sections also were used for determination of fiber areas. The areas of 50 type I and all type IIa fibers were measured in SOL by using a computer digitizing package. The areas of 50 type IIa and IIb fibers were measured in the PER, GAST, PLAN, and EDL. Because few type I fibers existed in the white portion of the GAST, and few type I fibers existed in the PER, EDL and PLAN, we digitized all type I fibers (25–50 fibers) in the photos obtained from these sections. Data Analysis We analyzed data by means of a 2 ⫻ 2 analysis of variance to determine effects of reduced hormone (Con vs Orx), treat-

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MUSCLE RESPONSE TO GONADECTOMY AND REDUCED USE, Brown Table 1: Body Mass and Muscle Wet Mass for SOL, PLAN, GAST, PER, and EDL, by Groups

Table 3: P0, P0/Muscle Mass, TPT, and Twitch RT1/2 Animal Group

Animal Group Con (n ⫽ 14)

Orx (n ⫽ 15)

Muscle

HLU (n ⫽ 9) Orx-HLU (n ⫽ 10)

Body weight (g) Initial 479 ⫾ 13 493 ⫾ 17 460 ⫾ 16 Final 486 ⫾ 13 496 ⫾ 18 438 ⫾ 18 Muscle wet mass (mg) SOL 209 ⫾ 7 214 ⫾ 6 156 ⫾ 6 PLAN 501 ⫾ 25 544 ⫾ 25 418 ⫾ 24 GAST 2688 ⫾ 120 2780 ⫾ 129 2097 ⫾ 105 PER 271 ⫾ 16 242 ⫾ 14 258 ⫾ 12 EDL 210 ⫾ 12 193 ⫾ 11 192 ⫾ 11

466 ⫾ 16 433 ⫾ 16

144 ⫾ 6 431 ⫾ 22 2093 ⫾ 109 238 ⫾ 13 186 ⫾ 11

Values are mean ⫾ standard error of the mean. Boldface values: main effect for hindlimb unloading (p ⬍ .05). No main effects existed for Orx or Orx ⫻ HLU interactions.

ment (Con vs HLU), or Orx ⫻ HLU interaction. Significance was set at p of .05 or less.

SOL P0 (g) P0/mass TPT (ms) RT1/2 (ms) PLAN P0 (g) P0/mass TPT (ms) RT1/2 (ms) PER P0 (g) P0/mass TPT (ms) RT1/2 (ms) EDL P0 (g) P0/mass TPT (ms) RT1/2 (ms)

Con

Orx

HLU

Orx-HLU

185 ⫾ 13 0.88 ⫾ 0.08 50.1 ⫾ 4.0 74.4 ⫾ 5.9

148 ⫾ 15* 0.70 ⫾ 0.09* 53.4 ⫾ 4.7 70.5 ⫾ 6.9

121 ⫾ 11 0.78 ⫾ 0.07 44.5 ⫾ 3.9 71.9 ⫾ 5.7

79 ⫾ 15* 0.56 ⫾ 0.09* 45.3 ⫾ 5.3 56.6 ⫾ 7.8

563 ⫾ 35 1.12 ⫾ 0.07 33.8 ⫾ 1.8 32.3 ⫾ 2.3

544 ⫾ 40 0.92 ⫾ 0.08* 26.9 ⫾ 2.0* 20.2 ⫾ 2.5*

498 ⫾ 31 1.21 ⫾ 0.07 36.5 ⫾ 1.6* 30.4 ⫾ 2.1

420 ⫾ 42 0.98 ⫾ 0.09* 25.8 ⫾ 2.2* 20.4 ⫾ 2.8*

445 ⫾ 30 1.67 ⫾ 0.11 37.0 ⫾ 1.6 36.2 ⫾ 2.5

316 ⫾ 41* 1.21 ⫾ 0.14* 28.6 ⫾ 2.0* 20.6 ⫾ 3.2*

462 ⫾ 29 1.78 ⫾ 0.11 39.4 ⫾ 1.6 34.1 ⫾ 2.5

384 ⫾ 38* 1.64 ⫾ 0.13* 27.1 ⫾ 2.0* 24.1 ⫾ 3.2*

324 ⫾ 20 1.58 ⫾ 0.09 29.0 ⫾ 1.4 23.5 ⫾ 2.2

251 ⫾ 24* 1.27 ⫾ 0.10* 24.4 ⫾ 1.5* 18.0 ⫾ 2.5*

337 ⫾ 18 1.76 ⫾ 0.08 32.1 ⫾ 1.3 28.6 ⫾ 2.0

278 ⫾ 25* 1.49 ⫾ 0.11* 25.6 ⫾ 1.7* 19.9 ⫾ 2.8*

Values are mean ⫾ standard error of the mean. Boldface values: main effects (p ⬍ .05) for unloading. * Main effects for Orx (p ⬍ .05).

RESULTS Body and Muscle Mass and Muscle Weight to Body Weight Ratios Orx. Gonadectomy had no effect on body weight or in muscle mass (table 1). Muscle weight to body weight ratios were not different for Con and Orx groups (table 2). HLU. Animals with unloaded hind legs had a significant decline in body weight ( p ⬍ .05). Unloading also resulted in a significant ( p ⬍ .05) loss of muscle weight in postural and locomotor musculature (SOL, PLAN, GAST), but had no effect on PER or EDL muscles. Orx ⴛ HLU. The decline in body weight and muscle mass for Orx-HLU animals was similar to that observed for unweighted animals without Orx. The ratios of muscle weight to body weight also were not different between HLU and OrxHLU groups. Contractile Characteristics Orx. The P0 of SOL, PER, and EDL declined significantly after Orx. Although Orx had no effect on skeletal muscle mass, we found a significant decline in muscle force/mass (specific tension) in all 4 muscles studied. The decline in specific tension

Table 2: Muscle Weight to Body Weight Ratios Animal Group Muscle

SOL PLAN GAST PER EDL

Con

Orx

HLU

Orx-HLU

0.43 ⫾ 0.03 1.07 ⫾ 0.05 0.55 ⫾ 0.23 0.55 ⫾ 0.04 0.43 ⫾ 0.03

0.43 ⫾ 0.04 1.11 ⫾ 0.05 0.56 ⫾ 0.24 0.50 ⫾ 0.05 0.39 ⫾ 0.03

0.36 ⫾ 0.04 0.96 ⫾ 0.05 0.48 ⫾ 0.24 0.59 ⫾ 0.05 0.44 ⫾ 0.03

0.33 ⫾ 0.03 1.00 ⫾ 0.04 0.48 ⫾ 0.33 0.55 ⫾ 0.04 0.43 ⫾ 0.03

Values are mean ⫾ standard error of the mean. Boldface values: main effect for HLU (p ⬍ .05).

ranged from 18% (PLAN) to 28% (PER) and did not appear to favor postural versus nonpostural muscles or muscles with a predominate fiber type. Contraction times generally were faster in Orx than Con rats (table 3). TPT was speeded up in Orx compared with Con animals for PLAN, PER, and EDL ( p ⬍ .05). RT1/2 also was shorter in Orx than Con animals for 3 of 4 muscles studied. HLU. Only in PLAN and SOL did P0 decline significantly with unloading, and we found a significant P0 increase with unloading in PER and EDL. Unloading had no apparent effect on contraction times, a finding consistent with previous studies for animals this age in our laboratories.10 Orx ⴛ HLU. In Orx animals, SOL specific tension was further reduced with unweighting. Although there were no Orx ⫻ HLU interactions, results for SOL-specific tension approached significance ( p ⫽ .064). Fiber-Type Distribution and Fiber Areas Orx. For PLAN, type IIa fiber percentages ⫾ standard error of the mean were 19.3% ⫾ 4.6%, 16.8% ⫾ 2.6%, 20.5% ⫾ 1.9%, and 23.2% ⫾ 1.9%, and type IIb fiber percentages were 68.2% ⫾ 6.1%, 76.3% ⫾ 3.1%, 68.7% ⫾ 2.4%, and 62% ⫾ 4.8% for Con, Orx, and Orx-HLU groups, respectively (main effect for gonadectomy, p ⬍ .05) (table 4). There were no apparent changes in fiber area after Orx. HLU. We found no hindlimb unloading effects on fibertype distribution. In GAST and SOL, all fiber types were atrophied in unloaded animals ( p ⬍ .05). Orx ⴛ HLU. There were no significant interactions. DISCUSSION The major findings of the present study are that: (1) Orx had little effect on muscle mass, but it significantly reduced muscle force, ie, strength; (2) HLU produced a similar degree of atrophy and loss of muscle force in gonadectomized and intact animals; and (3) contraction times (TPT, RT1/2) generally were faster in Orx rats than in the Con group. Results extend previArch Phys Med Rehabil Vol 82, January 2001

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MUSCLE RESPONSE TO GONADECTOMY AND REDUCED USE, Brown Table 4: Fiber Cross-Sectional Areas (␮m2)

Muscle

SOL I IIa PLAN I IIa IIb GAST I IIa IIb PER I IIa IIb EDL I IIa IIb

Con

Orx

HLU

Orx-HLU

4678 ⫾ 384 4192 ⫾ 599

4145 ⫾ 227 4060 ⫾ 154

2425 ⫾ 183 2574 ⫾ 192

2828 ⫾ 351 2419 ⫾ 790

2445 ⫾ 424 2398 ⫾ 331 3805 ⫾ 399

1735 ⫾ 125 2358 ⫾ 103 3708 ⫾ 227

1918 ⫾ 149 2251 ⫾ 180 3615 ⫾ 167

1615 ⫾ 59 2060 ⫾ 110 3213 ⫾ 191

1767 ⫾ 122 2295 ⫾ 224 3641 ⫾ 225

2276 ⫾ 129 2465 ⫾ 90 3537 ⫾ 121

1482 ⫾ 111 2074 ⫾ 224 3102 ⫾ 208

1500 ⫾ 169 1702 ⫾ 118 2874 ⫾ 226

1828 ⫾ 269 2128 ⫾ 345 3252 ⫾ 473

1524 ⫾ 96 2017 ⫾ 128 3515 ⫾ 247

1562 ⫾ 101 1930 ⫾ 124 2731 ⫾ 261

1377 ⫾ 75 1909 ⫾ 82 3142 ⫾ 93

1234 ⫾ 459 1204 ⫾ 165 2950 ⫾ 636

1055 ⫾ 134 1474 ⫾ 155 3164 ⫾ 384

1334 ⫾ 120 1557 ⫾ 84 2846 ⫾ 318

1065 ⫾ 96 1462 ⫾ 130 2637 ⫾ 229

Values are means ⫾ standard error of the mean. Boldface values: main effect for hindlimb unloading (p ⬍ .05).

ous observations and raise additional questions for study, particularly questions related to the mechanisms responsible for the change in specific muscle tension with Orx and unweighting. Few reports exist that highlight the effect of TST loss on contractile tension. Characteristically, a loss in muscle mass results in a reduction in muscle strength of similar magnitude,19 but that did not occur in the present investigation. Orx rats in the present study showed no change in muscle mass or fiber area, but did show a significant decline in contractile force. This finding suggests that (1) there is a dissociation between muscle strength and mass with the loss of TST, and (2) a breakdown (but not actual loss) of such contractile proteins as myosin may have occurred. This observation is consistent with the findings of Prezant et al.13 Examining the diaphragm in castrated rats, they found little effect of castration on muscle weight but specific force (force/cross-sectional area) was significantly less than that of controls. In the rats of Prezant, myosin heavy chain IIb was reduced, which was correlated with the loss of muscle force observed. Breakdown of myosin heavy chain may be the mechanism responsible for the loss of contractile force in the present study’s rats, but additional investigation is required to gain further evidence. The lack of change in muscle mass in our investigation was unexpected. Although Prezant13 did not observe a change in diaphragm muscle mass, other investigators2,5 have reported a decline in muscle size with hormone deficiency. Rats in our study were about 8 months of age when examined, a time when body weight and muscle weight are stable. Perhaps rapidly growing rats and boys and older declining men are more susceptible to muscle weight changes than mature adults. Although TST levels were not measured in the present investigation, it is difficult to conclude that TST levels were unaffected by Orx given the significant change in contractile function. Consistent with previous investigations,8-10 HLU resulted in atrophy in the locomotor and postural musculature of the calf and a decline in muscle force that was similar in magnitude to the decline in muscle mass. Findings suggest that total muscle Arch Phys Med Rehabil Vol 82, January 2001

protein and contractile protein were diminished. Circulating plasma levels of TST decline with unweighting,11,12 and lowered TST may have contributed to the decrease in contractile tension found in the present study. However, a similar magnitude of muscle atrophy and loss in contractile force occurs with unweighting in female rats.20 If circulating levels of TST influence the decline in muscle function in male rats, our findings may indicate a gender difference in response to unweighting. Possibly, a loss of mechanical stress and strain forces leads to the decline in muscle mass and strength with unweighting in male and female rats. Although we did not measure TST in the present investigation, we cannot conclude that TST levels were unaffected by Orx given the significant change in contractile function. The decline in specific tension (force/muscle mass) with Orx ranged from 18% to 27.5% in the 4 muscles examined physiologically, with no apparent predilection for any particular fiber-type dominance or muscle function (eg, postural vs nonpostural). This finding contrasts with hindlimb unloading, which clearly has a more detrimental effect on the postural and locomotor muscles such as SOL and GAST.10,17,20 The functional effects of TST, it seems, are evident in all muscle and fiber types. The present investigation and findings from other studies10,17 indicate that hindlimb unweighting lowers muscle mass and force in male rats. In the present study, Orx also resulted in a loss of muscle force generating capability. We expected that the combined effects of Orx and HLU would be more detrimental to muscle P0 than the independent effects of Orx and HLU, but results did not support this hypothesis. Perhaps the study was conducted over too short a time to detect the independent effects of hormone loss and reduced physical activity. Possibly, a decline in muscle force occurs at a certain pace, regardless of the number of influences contributing to the loss. We found no loss in fiber cross-sectional area with Orx in any of the 5 muscles we studied, which is consistent with the finding that none of the 5 muscles weighed less than those of controls. Two investigators,13,21 examining the diaphragm and caudofemoralis, found type IIb cross-sectional area to decrease with Orx; perhaps the hindlimb musculature is more resistant to specific fiber-type atrophy than those muscles. Possibly fiber size would decline in Orx of more than 4 weeks’ duration. In the present study, TPT for PLAN, PER, and EDL and RT1/2 in all 4 muscles were faster in Orx animals than in controls, whether unloaded or not. The TST-related change in contraction times was not explained by differences in fiber type as assessed by ATPase. Fiber typing as performed in the present study is probably not sensitive enough to detect subtle alterations in myosin heavy chain expression. ATPase staining is also not likely to account for the expression of multiple myosin heavy chain isoforms with a muscle fiber. Additional characterization of subtle isoform changes may be required to explain the change observed in contraction time. Also unclear is whether the faster TPT and RT1/2 have functional implications. Although a speeding of contraction times with HLU has not been seen in this laboratory,10,20 or in the laboratory of Takahashi et al,22 where animals older than 6 months of age were studied, it has been suggested that HLU increases sacroplasmic reticulum (SR) Ca2⫹ uptake speed by increasing the density of the SR Ca2⫹-ATPase or by increasing the proportion of fast Ca2⫹-ATPase.23 Because no differences existed in contraction time in Orx animals in the control or unloaded state, TST may also affect the SR Ca2⫹ uptake speed, but the mechanism of action is unclear.

MUSCLE RESPONSE TO GONADECTOMY AND REDUCED USE, Brown

The present findings complement those recently observed in humans by Zachwieja et al.24 In their study of men on 28 days of bed rest, lean mass was maintained in the 6 subjects receiving TST injections, but hormone injections had no effect on the decline in muscle strength. Controls and TST-injected men had the same magnitude of loss in strength in both the upper and lower extremities with bed rest, suggesting that different hormone-mediated mechanisms are responsible for the maintenance of muscle mass and contractile proteins. Hormone-mediated mechanisms may also be responsible for the dissociation between muscle force and mass in older men.24

11. 12.

13.

CONCLUSION Orx had no effect on muscle mass, but specific tension was significantly reduced in all muscles examined, and TPT and RT1/2 were faster in 3 of 4 muscles examined. The effects of HLU were similar in control and gonadectomized groups.

14.

Acknowledgment: Excellent technical assistance was provided by Sarah Friskey.

16.

References 1. Florini J. Effects of testosterone on qualitative pattern of protein synthesis in skeletal muscle. Biochemistry 1970;9:909-12. 2. Vermeulen A, Goemaere S, Kaufman JM. Testosterone, body composition and aging. J Endocrinol Invest 1999;22 Suppl 5:110-6. 3. Ameredes BR, Watchko JF, Daood MJ, Rosas JF, Donahoe MP, Rogers RM. Growth hormone improves body mass recovery with refeeding after chronic undernutrition-induced muscle atrophy in aging male rats. J Nutr 1999;129:2264-70. 4. Grindeland RE, Roy RR, Edgerton VR, Grossman EJ, Mukku VR, Jiang B, et al. Interactive effects of growth hormone and exercise on muscle mass in suspended rats. Am J Physiol 1994;267:R31622. 5. Sidor CA, Blackburn DG. Effects of testosterone administration and castration on the forelimb musculature of male leopard frogs, Rana pipiens. J Exp Zool 1998;280:28-37. 6. Bricout VA, Serrurier BD, Bigard AX, Guezennec CY. Effects of hindlimb suspension and androgen treatment on testosterone receptors in rat skeletal muscles. Eur J Appl Physiol 1999;79:443-8. 7. Morey-Hilton ER, Globus RK. Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight. Bone 1998;5(Suppl):83S-8S. 8. Musacchia XJ, Steffen JM, Fell RD, Dombrowski MJ. Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats. J Appl Physiol 1990;68:2248-53. 9. Winiarski AM, Roy RR, Alford EK, Chiang PC, Edgerton VR. Mechanical properties of rat skeletal muscle after hind limb suspension. Exp Neurol 1987;96:650-60. 10. Brown M, Hasser EM. Weight-bearing effects on skeletal muscle

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Suppliers a. Charles River Laboratories, Inc, 251 Ballardvale St, Wilmington, MA 01887. b. Astro-Med, Inc, 600 East Greenwich Ave, W Warwick, RI 02893.

Arch Phys Med Rehabil Vol 82, January 2001